PROJECT SUMMARY/ABSTRACT Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related expansion of hematopoietic stem cells that harbor somatic alterations without presenting other hematologic abnormalities. CHIP has been detected in normal peripheral blood of cancer patients with solid tumors and has been suggested to have a permissive role in therapy-related secondary myeloid disease development. Our recent analysis of clinical sequencing of 113,079 solid tumor specimens demonstrated that CHIP clones are also present in the solid tumor microenvironment due to admixed mutated hematopoietic elements; however, enrichment of admixed CHIP, its evolution under therapy, and its clinical impact on solid tumor treatment are poorly understood. In this proposal, we will characterize CHIP in the context of breast invasive carcinoma, which is treated with chemotherapeutics in both adjuvant and neoadjuvant settings and has been shown to have prognostic interactions with infiltrating hematopoietic cells in its microenvironment. We hypothesize that CHIP exhibits a distinct genomic landscape when enriched in breast tumor microenvironment, evolves under breast tumor treatment, and is correlated with the development of therapy-induced hematological toxicity. To test these hypotheses, we will assemble a cohort of 1,200 newly diagnosed breast cancer patients, collect comprehensive clinical data as well as sequential pre- and post-treatment peripheral blood and breast tumor samples, and profile CHIP at high resolution in three aims. First, we will determine the mutational spectrum of admixed CHIP before breast tumor treatment. Using high-depth sequencing of peripheral blood and breast tumor samples, we will detect CHIP mutations at >0.1% allele frequency and correlate the prevalence of admixed CHIP with the level of infiltrating lymphocytes and other hematopoietic markers. Using single-cell genomic analysis, we will resolve the number of exclusive CHIP clones. Second, we will profile CHIP after chemotherapy in peripheral blood ? and breast tumor samples in neoadjuvant settings ? to study its evolution by assessing mutation-specific fitness and therapeutic bottleneck size, using hormonal therapy as control. We will also investigate the effect of granulocyte-colony stimulation on CHIP's clonal dynamics. Longitudinal peripheral blood sampling will elucidate the long-term evolution of CHIP 1-2 years after the end of breast tumor's treatment. Third, we will develop a statistical regression model to determine the distinct CHIP clones that may be correlated with clinical response and development of therapy-induced hematological toxicity. This study is novel in its utilization of systematically collected clinical and high-resolution molecular data, and it will provide insight on CHIP's clonal evolution under breast tumor treatment. Moreover, it will illustrate the significance of molecularly defined clonal analysis of hematopoietic populations as a fundamental predictor of therapy-induced hematological complications. Finally, it will establish a platform for long-term clinical and molecular inquiries of CHIP's progression to therapy-related myeloid neoplasms in high-risk patients.